Emily C. Lennert
DNA, adhesive, tape, lift, clothing, sampling, yield, polymerase chain reaction, PCR, electropherogram
- Forsberg, C.; Jansson, L.; Ansell, R.; Hedman, J. High-throughput DNA extraction of forensic adhesive tapes. Forensic Science International: Genetics. 2016, 24, 158-163.
- Walsh, P. S.; Metzger, D. A.; Higuchi, R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques. 1991, 10 (4), 506-513.
The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.
Tape-lifting is a well-established method for collection of biological evidence, and can efficiently collect cells and DNA to allow for subsequent analysis. Previous studies cited by the authors have shown higher yields of DNA evidence recovered by tape lifts compared to swabbing on certain fabrics. The sampling procedure is simple: tape is pressed against the sample surface several times to collect a sample. The subsequent DNA extraction procedure is more complex, due to the rigidity, size, and adhesives that are characteristic of the sampling matrix. Previously presented methods for DNA extraction require a fair amount of manual labor, and include swabbing of the tape and subsequent swab extraction, extraction of the tape within an extraction buffer followed by DNA extraction from the buffer, and more. The authors presented an extraction procedure requiring limited manual labor for removal of DNA from adhesive tapes. This research was conducted by a forensic laboratory, and was later implemented in the lab’s operating procedures.
SceneSafe FAST tape lifts were taken from various items of clothing worn by volunteers. The clothes used as reference material were long sleeve t-shirts or button-down shirts, which were washed prior to use and worn for two office work days, approximately 30 hours. These reference samples were used to compare proposed extraction methods to the in-house method and to evaluate DNA yield for each. Reference DNA profiles were collected from volunteers to allow for known references and to ensure that the DNA profiles obtained during analysis originated from the volunteer. Additional samples were collected after normal wear and analyzed via the lab’s previously established, in-house casework method as well as the proposed extraction methods. These additional samples were caps, gloves, and sweaters worn by volunteers; 10 of each item were collected and tape lifts using the in-house tape and the SceneSafe FAST tape. These samples were used to compare the in-house method to the developed methods, as well as to evaluate the effect of different fabric types on DNA recovery. Blank samples were collected prior to wear for each item as well.
DNA Extraction Methods
Four extraction methods were examined. First, the in-house method was examined. Two methods using direct lysis, i.e. breakdown through rupture of the cell membrane, with slight differences in extraction buffer, were developed and examined. A fourth method was also evaluated; this method was a commercially available extraction kit, PrepFiler Express BTA Forensic DNA Extraction kit on Automate Express.
The in-house method used was a Chelex based standard method described in previous studies. Two Chelex stock solutions, 5% and 20% Chelex in water, are used to extract DNA from cut pieces of tape.
Developed Extraction Method 1: Direct Lysis with Chelex
By this method, a lysis buffer of 5% Chelex solution with 0.2 Tween 20 and 0.1 mg Proteinase K was used. Each tape was placed in a tube and 1 mL of lysis buffer was added. After 30 min at room temperature and vortexing, i.e. mixing, the sample underwent 2 incubation periods. A 45 min incubation at 56 ˚C, followed by vortexing and another incubation at 100 ˚C for 20 min was performed. Sample volumes were then reduced to 200 μL.
Developed Extraction Method 2: Direct Lysis with TE Buffer
By this method, a lysis buffer of TE buffer, consisting of 10 mM Tris and 0.1 mM EDTA at pH 8, with 0.2 Tween 20 and 0.1 mg Proteinase K was used. Each tape was placed in a tube and 1 mL of lysis buffer was added. After 30 min at room temperature and vortexing, i.e. mixing, the sample underwent 2 incubation periods. A 45 min incubation at 56 ˚C, followed by vortexing and another incubation at 100 ˚C for 20 min was performed. Sample volumes were then reduced to 200 μL.
PrepFiler Express BTA/Automate Express Method
User guidelines were followed for the extraction kit. Some modifications were made: the volume of extraction buffer was increased from 230μL to 500 μL to ensure that the tape was completely covered, the lysis products were run longer, and the final volume was adjusted to 200 μL to match that of the other methods.
Evaluation of Methods
Following extraction, the resulting DNA extracts were evaluated for extraction efficiency by quantitative polymerase chain reaction (PCR) using a quantification kit on a PCR system. Each sample was normalized prior to amplification by using 0.5 ng of DNA, according to manufacturer recommendations.
The PrepFiler Express BTA/Automate Express method was compared to the developed direct lysis methods using data from the reference material samples, i.e. t-shirts and button-downs. The direct lysis method with Chelex showed higher DNA yield, measured in ng/μL, compared to the PrepFiler method; however, the differences were not deemed statistically significant. Direct lysis with TE buffer produced significantly higher DNA yield than PrepFiler for t-shirts; however, DNA yield for button-downs from direct lysis with TE buffer was low. The authors state that the uneven performance of the TE buffer method may be due to the chelating strength of the EDTA. Chelating agents, like EDTA and Chelex, are compounds that form several bonds with a single metal ion. In DNA extraction, this chelation serves as a cofactor for nucleases, which are enzymes that play a role in DNA stability. Consequently, a more efficient chelating agent will result in a more stable DNA extract. EDTA is a weaker chelator than Chelex, which may account for the TE buffer’s inconsistent performance between samples.
The in-house Chelex method was then compared to the direct lysis Chelex method. DNA yield, reproducibility, DNA extract stability, and contamination risk were evaluated. Reference materials, t-shirts and button-downs, were examined first. Although direct lysis produced higher DNA yields than the in-house method, the differences were not deemed statistically significant. Complete electropherograms, a form of output from DNA sequencing by capillary electrophoresis, were produced for DNA extracts from each method. Caps, gloves, and sweaters, the items denoted as normally worn clothing, were then evaluated. Sweaters were sampled in two places: on the sleeve and at the collar. Sleeve, glove, and cap samples showed higher DNA yield for direct lysis with Chelex. Only collar samples produced higher DNA yield by the in-house method. Statistical analysis revealed that the differences in DNA yield between the methods were not statistically significant. No differences in reproducibility were observed. DNA extracts for both methods exhibited good stability, with no effect on electropherogram quality or DNA yield after 2 weeks storage at 8 ˚C. Negative controls analyzed by the direct lysis method produced blank results, indicating low risk of DNA contamination in the method. The laboratory considered the method suitable for casework and implemented the direct lysis Chelex method in September 2015.
After switching to the direct lysis Chelex method and SceneSafe FAST tape for lifting, the lab determined that the percentage of cases with a suitable amount of DNA for analysis increased from 71% to 76%. Profiles obtained from the DNA also improved, with the fraction of single-donor profiles and clear major profiles increasing from 20% to 26%. Recovery of DNA mixtures is a common problem in tape-lifting, due to the ease of transfer of cells and touch DNA.The authors reported the throughput of cases by each method as well, as seen in table 1 within the study. Comparing the lab’s previous in-house Chelex operating procedure to the more recently implemented direct lysis Chelex procedure, less manual effort was required of the analyst. By the old in-house Chelex method, a maximum of 50 samples could be processed per day by each analyst. With the direct lysis Chelex method, a maximum of 120 samples could be processed in the same amount of time. The cost per extraction did not differ between the methods.
- The direct lysis Chelex method was determined to perform as well or better than the in-house Chelex method that was being used. As a result, the lab implemented the direct lysis method for casework in September 2015.
- The direct lysis Chelex method allows for high-throughput analysis. A much larger quantity of samples can be analyzed per day, 120, compared to the old Chelex method, 50, with no increase in cost.
High-throughput methods are desirable to prevent backlogs from forming. However, method quality must not be sacrificed in the quest for more rapid analysis methods, and it is desirable to keep expenses from rising.
- 22The direct lysis Chelex method allows for high throughput analysis of tape-lift DNA samples without affecting DNA yield or electropherogram quality.